[Abstract] Understanding the mechanisms underlying memory formation is a fundamental focus in neuroscience. As a potential molecular and cellular basis of synapse plasticity, including memory formation, we have proposed a novel hypothesis, the local feedback model (Yoshihara et al., 2005, Science 310: 858-863). In this model, we postulate that reciprocal strengthening of presynaptic and postsynaptic signals by a positive feedback loop at single synapses keeps individual synapses potentiated, leading to eventual morphological changes and perpetually strengthened synapses. This long-lasting change could form the basis by which the brain stores memories. At neuromuscular junctions (NMJs) of Drosophila embryos, we demonstrated that stimulating the motor axon at high frequency (100 Hz) induces a large (100-fold) prolonged increase in miniature release frequency. We have termed this phenomenon High Frequency Stimulation-induced Miniature Release (HFMR). Several lines of evidence suggest that HFMR and synaptic growth require local postsynaptic retrograde signaling, mediated by a postsynaptic Ca2+ sensor, Synaptogtagmin 4 (Syt 4). A key prediction of the local feedback hypothesis is that the acute synaptic plasticity revealed by HFMR is translated into long-term structural changes. To fully test this prediction, we have begun to establish a whole- embryo culture system in which synaptic growth will be visualized in real time while stimulating motor nerves and recording from the muscles. The goal of this proposal is to test important predictions of the local feedback hypothesis, and to understand the molecular and cellular mechanism of this process. In particular, we have shown that postsynaptic Ca2+ is essential for retrograde signaling and we have evidence that Syt 4 functions as a Ca2+ sensor for the release of the retrograde signal. The goals of this project will be accomplished in three aims. (1) We will determine the signaling pathways responsible for the acute plasticity, including describing the role of postsynaptic Ca2+ and identification of the retrograde signal. (2) We will examine a postsynaptic vesicle trafficking mechanism dependent on Synaptotagmin 4 as a postsynaptic Ca2+ sensor. (3) We will elucidate the molecular mechanisms of the transition from acute plasticity to morphological changes using a genetic analysis. We anticipate that the outcome of this study will shed considerable light on basic principles of memory mechanisms, and provide a framework to address memory-related diseases such as amnesia, dementia, and Alzheimer disease.